The Development of TGR5 Antagonists for the Treatment of Cholangiopathies

项目总结 这项建议的目标是开发胆汁酸受体TGR5的拮抗剂，作为一种潜在的治疗方法 对于多囊肝病(PLD)，一种以肝脏囊变为特征的遗传性胆管病。 胆管病是一组肝脏疾病，其中胆管细胞、上皮细胞排列在肝内胆汁内 管道是主要目标。PLD是不治之症，以孤立的常染色体显性PLD形式存在或与 常染色体显性遗传性多囊肾病和常染色体隐性遗传性多囊肾病。尽管最近 关于PLD发病机制的研究进展，目前还没有针对这些破坏性疾病的治疗方法。我们之前的工作 牵连的cAMP(在囊性胆管细胞中增加)作为一种重要的中枢成分 PLD中的信号通路失调，并引导我们采取cAMP靶向治疗疾病的策略。我们 TGR5是一种与cAMP信号相关的G蛋白偶联胆汁酸受体，在胆汁中起着重要的作用 CAMP在PLD肝脏囊变中的作用。我们发现TGR5在囊性癌中过度表达。 在体外和体内，发现TGR5激动剂增加细胞内cAMP，触发 胆管细胞在体外和PCK大鼠体内的过度增殖和囊性生长(病情恶化 进展)。进一步的研究结果支持TGR5‘S在肝囊性变发病机制中的作用。因此，我们的 目的是开发拮抗剂来抑制TGR5的活性，从而减少PLD的肝脏囊变。作为否 选择性小分子TGR5拮抗剂已有报道，我们完成了HTS鉴定了几个 前景看好的线索包括SBI-319。我们建议开展这一领先的优化活动，以开发口服可用药 用于体内测试的化合物，以验证TGR5作为PLD的潜在靶点。 中心假说。选择性的TGR5拮抗剂将抑制囊性胆管细胞内cAMP水平，从而减少 CAMP驱动的细胞增殖和肝脏囊变，为PLD提供了一个新的治疗靶点。去探索 我们的中心假设，我们将实现以下具体目标：目标1.设计和合成TGR5 活体内口服有效的拮抗剂。通过反复的化学循环，我们将确定适合于 体内药效研究。目的2.阐明SBI-319类似物在PLD中的作用机制。我们会1) 评估SBI-319类似物对：(I)囊性胆管细胞产生cAMP；(Ii)胆管细胞增殖； (3)3D培养中肝囊性结构的生长；2)检测TGR5和GαS蛋白的表达， 以及它们在用SBI-319处理后的偶联情况。目的3.评价TGR5拮抗剂作为一种 PLD的治疗。我们将评估SBI-319类似物对肝脏和肾脏囊变的影响，并阐明 抑制TGR5在体内疾病进展中的作用。 除了PLD作为一种潜在的治疗应用外，研究发现TGR5在 在非胆道癌(乳腺癌、胃癌、胰腺癌)中表达上调。发展中的 有效的TGR5拮抗剂很可能在PLD之外具有治疗应用。

PROJECT SUMMARY The objective of this proposal is to develop antagonists of the bile acid receptor TGR5 as a potential treatment for polycystic liver disease (PLD), a genetic cholangiopathy characterized by hepatic cystogenesis. Cholangiopathies are a group of liver diseases in which cholangiocytes, the epithelial cells lining intrahepatic bile ducts, are the primary target. PLD, is incurable and exists as isolated Autosomal Dominant PLD or co-exists with Autosomal Dominant Polycystic Kidney Disease (ADPKD) and Autosomal Recessive PKD. Despite recent advances in the pathogenesis of PLD, there are no therapies for these devastating conditions. Our previous work implicated cAMP (increased in cystic cholangiocytes) as an important central component in the network of dysregulated signaling pathways in PLD and led us to cAMP-targeted strategies for disease treatment. We discovered TGR5, a G protein-coupled bile acid receptor (GPCR) linked to cAMP signaling, plays an important role in cAMP-driven hepatic cystogenesis in PLD. We demonstrated that TGR5 is overexpressed in cystic cholangiocytes in vitro and in vivo, and found TGR5 agonists increase intracellular cAMP, triggering cholangiocyte hyper-proliferation and enhancing cyst growth both in vitro and in PCK rats (worsening disease progression). Further findings support TGR5's role in the pathogenesis of hepatic cystogenesis. Thus, our objective is to develop antagonists to inhibit TGR5 activity, thus reducing hepatic cystogenesis in PLD. As no selective small molecule TGR5 antagonists have been reported, we completed an HTS identifying several promising leads including SBI-319. We propose this lead optimization campaign to develop orally available compounds for in vivo testing to validate TGR5 as a potential target for PLD. Central Hypothesis. Selective TGR5 antagonists will inhibit cAMP levels in cystic cholangiocytes, thus reducing cAMP-driven cell proliferation and hepatic cystogenesis, yielding a novel therapeutic target for PLD. To explore our central hypothesis, we will perform the following specific aims: Aim 1. To design and synthesize TGR5 antagonists orally active in vivo. Through iterative cycles of chemistry, we will identify compounds suitable for in vivo efficacy studies. Aim 2. To clarify the mechanisms of action of SBI-319 analogs in PLD. We will 1) assess SBI-319 analog effects on: (i) cAMP production in cystic cholangiocytes; (ii) cholangiocyte proliferation; and (iii) growth of hepatic cystic structures in 3D cultures; 2) examine the expression of TGR5 and Gαs proteins, and their coupling upon treatment with SBI-319. Aim 3. To evaluate the efficacy of TGR5 antagonists as a treatment for PLD. We will assess effects of SBI-319 analogs on hepatic and renal cystogenesis and clarify the role of TGR5 inhibition in disease progression in vivo. In addition to PLD as a potential therapeutic application, findings show TGR5 is over-expressed in cholangiocarcinoma and is up-regulated in non-biliary cancers (breast, gastric, pancreas). Development of effective TGR5 antagonists would likely have therapeutic application beyond PLD.